Various processes have been used in the past to isolate valuable water insoluble commercial compounds from fermentation broths. Traditional technologies for isolating such compounds employ solid-liquid separations (e.g. filtration, centrifugation, etc.) to isolate the water-insoluble active ingredients and subsequent solid-liquid extractions to recover the activities. For example, the U.S. Pat. Nos. 4,117,118 and 4,215,199 to H arri describe processes for isolating cyclosporins A and B from fermentation broths involving the steps of centrifugation, homogenization, and multiple extractions (using methanol, ethylene chloride, and other water-immiscible organic solvents) with corresponding evaporations (i.e. concentrations). Thereafter, the final extracts are subjected to chromatographic purifications using silica gel and SEPHADEX.RTM. LH20 packings. Similar procedures are employed for isolating other types of water insoluble compounds such as lovastatin (an antihypercholesterolemic) and tacrolimus (FK-506, an immunosuppressant). Although these methodologies are currently used for industrial scale fermentations, they typically require expensive solid-liquid separators and solvent extractors/evaporators/condensers having high energy requirements. In addition, the product recovery yields from such processes are low due to multi-stage operations. Thus, capital investment and subsequent production costs are high.
As another example, the patent to Rudat (U.S. Pat. No. 5,256,547) describes a process for the production and isolation of cyclosporin A which involves mixing the culture with a filter aid such as recrystallized gypsum or calcite meal to form a suspension and filtering the mixture to obtain a moist biomass. The biomass is then dried and extracted two or three times with a lower carboxylic acid ester, or alternatively with a supercritical gas such as carbon dioxide. The extract is then defatted and chromatographed by preparative HPLC using silica gel or alumina oxide. This method offers limited advantages over those disclosed in earlier patents because it still suffers from multiple, complicated, and expensive operations.
Although the use of either microfiltration (M F) or ultrafiltration (UF) to clarify/filter aqueous fermentation broths has been established in the literature, extractions with organic solvents are usually performed as a secondary purification step to recover the active product. As noted above, the conventional purification procedures involve two distinct unit operations, namely, separation and extraction/evaporation. Generally for water insoluble products, the compound is first isolated from the large volume of aqueous fermentation broth and then purified by repeatedly extracting the compound with solvent and evaporating the solvent, so that the compound can be further extracted with a different solvent and evaporated until a concentration is achieved from which ultimate purification can take place. The repeated extractions and evaporations however, render the process very costly for large scale manufacture.
A unique feature of the present invention is in having a continuous processing system that obviates the need for separate extraction and evaporation steps after the initial centrifugation and/or filtration step. This technology offers many advantages over the prior art processes, including simplicity of design, reduced capital and manufacturing costs, and increased recovery yield. Furthermore, unlike the traditional processes, the entire process of the present invention is both automatable and fully contained which reduces both personnel and environmental exposure to the compound. This is an important consideration in that immunosuppressants and other potent therapeutic compounds may be highly toxic.